Molecular detection of sub-microscopic infections and Plasmodium falciparum histidine-rich protein-2 and 3 gene deletions in pre-elimination settings of South Africa

South Africa’s efforts toward eliminating malaria have positioned the country in the pre-elimination stage. Imported and sub-microscopic cases still contribute to the persistence of malaria in regions of low transmission as identified in this study where diagnostics is built largely on the use of Rapid Diagnostic Test (RDT). However, the presence of Pfhrp2/3 gene deletion is known to interfere with the accuracy of diagnosis with the use of RDT. Malaria elimination and detection of Pfhrp2/3 gene deletion in the pre-elimination setting requires accurate molecular surveillance. With the core objective of this study being the determination of the presence sub-microscopic malaria cases and deleted Pfhrp2/3 gene markers, a total of 354 samples were collected from five districts of KwaZulu Natal, South Africa. These samples were prepared for molecular analysis using primers and PCR conditions specific for amplification of 18S rRNA and msp-1gene. Positive amplicons were analysed for the presence of Pfhrp2/3 and flanking genes, along with Sanger sequencing and phylogenetic studies. Out of 354 samples collected 339 were tested negative with PfHRP2 based RDTs. Of these Pfhrp2 and Pfhrp3 gene deletions were confirmed in 94.7% (18/19) and 100% (19/19) respectively. High migration rate (75%) among the study participants was noted and phylogenetic analysis of sequenced isolates showed close evolutionary relatedness with India, United Kingdom, Iran, and Myanmar and China isolates. Molecular-based test is recommended as an essential surveillance tool for malaria management programs as the target focuses on elimination.

of Plasmodium falciparum are common in many endemic populations, particularly in low-transmission areas aiming for elimination 8 .These infections are of particular relevance to elimination programs, as they can persist for a number of months without any symptoms that would prompt seeking treatment 7,8 .While light microscopy examination of blood slides is the main method of detecting malaria infection, it has limited sensitivity and more sensitive methods such as polymerase chain reaction (PCR) are needed to detect sub-microscopic infections 8 .
According to Raman et al. 9 , detection of sub-microscopic (sub-patent) infections in South Africa communities is one of the key challenges that need to be addressed for South Africa to drive closer and ultimately achieve the desired elimination status,especially since these have the potential for further infection transmissibility 10 .Hermsen et al. 11 defined sub-microscopic infections as "low-density Plasmodium infections detected only by molecular methods" and it has been demonstrated that sub-microscopic cases are identified in greater number among adults in low-endemic settings, similar to what is found in South Africa 8,12 .Prevalence of sub-microscopic infections 13 in low malaria transmission areas has also been identified as serious threat in malaria elimination programmes 14,15 .However, core to the issue of sub-microscopic case detection and malaria elimination is the diagnostic approach.Malaria diagnostic method in South Africa is largely built around use of RDT.The operational principle of RDT as a malaria diagnostic tool is based on the identification of the antigens lactate dehydrogenase (LDH), aldolase or histidine-rich proteins in the parasite.LDH and aldolase antigens have been identified in all species of malaria while histidine-rich protein is specific for Plasmodium falciparum 16 .Deletion of the Pfhrp gene can lead to false negative reporting of RDT tests, of which Pfhrp2 and Pfhrp3 gene deletion has been widely reported in South America but less in Africa and Asia 17 , Irene 18 .
In preparation for the elimination stage, Solomon Islands, made use of a molecular based diagnostic method for the identification of sub-microscopic cases in one of the provinces.It effectively assisted with identifying underlining prevalence based on sub-microscopic infections and gave direction on approaching the elimination target 19 .An appropriate focus of resources and the drive to identify and deal with sub-microscopic infections as well as false negative RDT arising from deleted Pfhrp genes become important in view of the elimination target which has been set for 2025.We propose that in low transmission settings, false negative rapid diagnostic tests may contribute to an increase in sub-microscopic malaria infections.In addition, we hypothesize that the presence of Pfhrp2/3 gene deletions has a direct correlation with sub-microscopic malaria.This study therefore aims to conduct a molecular assessment of sub-microscopic infections and also determine the prevalence of deleted Pfhrp2 and Pfhrp3 genes in low transmission districts of Kwa Zulu Natal province in South Africa using RDT and conventional PCR.

The outcome of molecular tests
The nested reaction was used to screen for the 18S rRNA gene amplification among the RDT negative cassettes and it showed 9.4% (32/339) to be positive for P. falciparum.However, following a further analysis with Sanger sequencing, 5.9% (20/339) were noted to have a good chromatogram appearance.After subjecting these 20 samples to quality test by screening for msp1, 5.6% (19/339) of the samples were found suitable.

Discussion
Malaria RDT serves as a major point-of-care (POC) diagnostic tool in South Africa, and it is widely used in many parts of the country due to ease of application.However, limitations such as false negativity is associated with RDT use, hence, the use of microscopy as an adjunct tool in confirmation of a positive diagnosis of malaria.Although reliable, the accuracy of microscopy is dependent on a lot of factors which include availability of trained individuals, quality and the sensitivity of microscope used as well as the sample handling technique.In this study which was conducted in a malaria low transmission setting, we identified RDT positivity of 4.2% (15/354).Previous study in the province reported a prevalence rate of 2% for local cases 6 .Our finding aligns with the trend reported in low transmission settings where malaria positivity decreases as the elimination stage sets in Laban et al. 20 .By inference, this is a possible reflection of the effectiveness of the different methods that have been applied during the malaria elimination campaigns in the province.It could also be a reflection of regular improvement in the quality control methods.
No doubt, microscopy detection of plasmodium is a widely accepted "gold standard" for malaria diagnosis, but it is unable to detect low levels of the parasite, known as sub-microscopic infections 21 .However, more advanced molecular techniques such as polymerase chain reaction (PCR) are capable of detecting these infections with greater sensitivity, making them more suitable for routine diagnosis 21,22 .Another study suggested that nucleic acid methods are typically the most effective diagnostic tools for identifying Plasmodium species 23 .
Considering the low transmission setting of the study, we chose to use a more sensitive test as prescribed in the recommendation-3 of the World Health Organization Evidence Review Group on Malaria diagnosis in Low Transmission Settings 24 , by confirming the presence of the P. falciparum using the conventional PCR followed by Sanger sequencing of the positive amplicons.Following the analysis of the sub-microscopic infections, 9.4% of the negative RDT samples tested positive with conventional PCR, thus confirming the inherent shortfalls with the use of RDT as surveillance diagnostic tool in settings that are nearing elimination 25,26 .This justifies the call by the World Health Organization for the use of molecular tool during surveillance in pre-and post-elimination settings 27 .In fact, a study conducted in low transmission setting of Swaziland (eSwatini) by Ranadiwe et al. 26 affirmed the low sensitivity of RDT.Thus, as a country that looks forward to eliminating malaria, a practical step in a positive direction requires deploying a more efficient tool that can detect sexual and asexual stages of the parasites, particularly for surveillance in very low transmission areas.Molecular tools that operate on the principle of nucleic acid detection have been demonstrated to be efficient in this regard; an example of this is Loop-mediated isothermal amplification (LAMP).LAMP operates on the principle of gene amplification, but it is less complex when compared with conventional or real time PCR 24,28 .This relative advantage makes it a tool for consideration in large scale surveillance that is focused on identification of sub-microscopic cases,and this can be combined effectively with focal mass drug administrative program.
Although South Africa is designated as low transmission, imported cases from neighbouring countries such as Mozambique and Zimbabwe 29 are still common.Some of our study participants also had a travel history outside  2) indicates interdistrict, inter-province and international movement accounted for 64.6%, 8.3% and 2.1% respectively.Studies have shown that migration has the potential for spreading malaria [30][31][32] .There is the possibility that the identified sub-microscopic cases were actually spreading from rural to urban and vice-versa.In that regard, there is need to put in place measures that will curtail this spread among locations that have been certified as malaria-free and thus calling for priority into investing in research and strengthening of already existing elimination approach.
Based on the result obtained in the positive samples from the sub-microscopic cases, we built a Maximum-Likelihood phylogenetic tree to determine how genetically related and diverse are the isolates; and found close evolutionary and possible common ancestry between one of our P. falciparum isolates (OP341871.1)and those of Asian origin.Despite this close relatedness, the diversity in the local population was obvious.Genetic diversity has been determined in P. falciparum using different markers including the merozoite surface protein (msp) genes 33 and related to transmission in different environments.Abdelraheem et al. 34 reported a high genetic diversity among Plasmodium that were imported from different countries and implicated India as major importer of Plasmodium viva.According to Xu et al. 35 there was high density in P. falciparum isolates among returning Chinese migrant workers from Africa, thus supporting the claim of parasite exchange between the two continents of Africa and Asia.Although China has recently received a WHO certification of malaria elimination, there is a claim that imported malaria cases are often by Chinese workers in South Africa (and other parts of Africa) and these serve as vessels for the importation of the malaria parasite 36,37 .These might explain the close genetic relatedness in our P. falciparum isolates and those from China and India.
Since the initial identification of Phrp2/3 gene deletion in Peru 38 and the subsequent reports by many countries 39,40 , the growing concerns call for a widespread study in both high and low-transmission settings.In general, most documented reports are mainly studies that were carried out in regions that are malaria endemic as obtained in sub-Saharan Africa, Asia and South America 16,41 .Our study identified non-amplification of a large proportion of the Pfhrp2 and the Pfhrp3 genes − 94.7% and 100% respectively.This finding is in tandem with the outcome of previous studies that were carried out in places with a low prevalence of malaria (less than 1 per 1000 population) like Honduras (Pfhrp2-0%, Pfhrp3-96.2%)and Guatemala (Pfhrp2-14.3%,Pfhrp3-90.5%)which recorded high gene deletions, especially the Pfhrp3 gene 38,39 .Also, a meta-analysed mean prevalence by Nyataya et al. 16 showed that there is a higher mean incidence of Pfhrp3 in comparison with Pfhrp2.The documented African studies were mainly from the malaria-endemic countries in the sub-Saharan region and no study has been documented so far from the countries where the malaria endemic level is low 41 .To the best knowledge of the authors, this is the first study in a low endemic region located in sub-Saharan Africa.According to Berzosa et al. 42 , there is a possibility of the selection of Pfhrp2 gene deletion with the over-use of Hrp-based RDTs.The identification of a large proportion of the Pfhrp 2/3 gene deletion in places which have been regarded as almost free of malaria transmission makes a pointer to the need for a closer look into the surveillance diagnostic methods that are currently being applied.
Low malaria parasite level is another reason associated with false-negative RDT, especially in low endemicity settings 43 .To further shed light on this, study by Beshir et al. 44 in which the relationship between the parasitemia level and the gene deletion was modelled, it was noted that "the probability of RDT positivity for Pfhrp2 negative samples in the presence of Pfhrp3 samples increased with parasitaemia and was close to the one for Pfhrp2 positive samples in the presence of Pfhrp3 positive samples for parasitaemia levels > 1000 μl.Such proximity of the two probabilities together with the reduced number of Pfhrp2 negative samples in the presence of Pfhrp3 amplified samples implied that they were not statistically different as a function of parasitaemia".Bharti et al. 45 further reported that 48% of subjects with high parasite density tested negative with RDT, thus indicating that an exclusively low parasitemia is not enough to explain false negative RDT.This is also in line with the report by Wurtz et al. 46 who identified no difference between the group with deletion and those without deletion.Although parasitemia level was not evaluated in this study it may not have a significant impact on the noted outcomes since good concentration (1.1-2.9 ng/µl) as well as quality DNA was ensured in the positive samples that were used for the final amplification step.
Part of the study limitation identified include the fact that even though the authors used the World Health Organization recommendation of molecular based diagnostic method in the low transmission setting, the use of microscopy could still be considered in future studies.However, the number of trained personnel in malaria microscopy has fallen drastically in the past few years in South Africa thus posing challenges to microscopy-based diagnosis.Furthermore, since this study was done using five KwaZulu Natal districts, it will be worthwhile if a national survey be conducted to give the overall picture and prevalence rate of both sub-microscopic cases and deleted Pfhrp2/3 gene.This will assist with national malaria program planning as well as ways of recommendations as per World Health Organization policy.
In conclusion, the study identified the presence of sub-microscopic P. falciparum infections Pfhrp2/3 gene deletions in KwaZulu Natal, South Africa where malaria is in low transmission.The positivity rates obtained in both instances were in consonance with that found in low transmission zones and are significant at a time when the malaria elimination agenda is looking forward to 2025.A high rate of migration also characterized the study with the phylogenetic analysis suggesting a possible evolutionary connection between the strain from South Africa and that of China United Kingdom, Iran, Myanmar and India.Having invested so much in machinery and manpower to reach the pre-elimination status, our study findings are indications that South Africa needs to consider a national study on the prevalence of deleted Pfhrp2/3 genes.In addition, bearing in mind the possibility of the establishment of cycles of infection that could result from sub-microscopic residuals in the presence of appropriate conditions, intensifying efforts to achieve elimination must not undermine consideration of an application of molecular-based diagnostics which have greater potential to overcome the limitations of RDT and microscopy and also provide more usefulness in the disease surveillance.

Method Study area
The study was conducted in KwaZulu Natal which is known for malaria transmission where Plasmodium falciparum is the predominant species (Fig. 1).It is located on the coordinates 28.5306°S, 30.8958°E with warm and subtropics climates and a land mass of 94,000km 2 with eleven districts municipalities which are mostly rural, and harbour a population of about 11.1 million.The province is among the three in the country that still records very-low to a low incidence of malaria cases.

Study population
Samples were collected in the following districts of KwaZulu Natal: Ethekwini, Ilembe, Ugu, Uthungulu, and Umkhumyakude (Fig. 1).The health facilities are situated in rural areas, with the exception of the Ethekwini district, where they are situated in urban areas.The study received ethical approval from the Biomedical Research Ethics committee of the University of KwaZulu Natal South Africa (BREC/00001815/2020). Participants with symptoms suggestive of malaria were recruited from health facilities in the listed districts after obtaining informed consents from interested individuals.Accompanied minors are allowed to participate following parental or guardians' informed consents.Individuals with history suggestive of bleeding disorder were excluded.All methods were carried out following relevant guidelines and regulations.

Sample size determination
Since there were more than 10,000 participants in the study, the sample size was determined using Fisher's formula 47 .Assuming a 95% confidence interval is equal to 1.96, a 5% acceptable margin of error, and a maximum variability of 50% given the unknown prevalence, one can estimate the prevalence of the desired outcomes.384 patients will be needed as a sample size based on the statistical parameters combined.Each medical facility's patients were purposefully chosen until the calculated sample size was reached for each facility.The power of the sample (1 − β) and the (%) chance of detecting differences in the study were set at 80%.However, out of 384 participants approached, only 354 accepted to participate making it a 92.2% response rate bearing in mind that "a response rate of ≥ 80% was expected" 48 .
Furthermore, the study used both purposive and convenient sampling to select people who agreed to participate in the survey.According to Teddlie and Yu 49 and Radhakrishnan 50 .Convenience sampling involves selecting samples that are both readily available and willing to participate in a study, whereas purposive sampling is predicated on the idea that researcher knowledge of the population can be used to select sample members.The sampling period for our study was from March 2021 to January 2022 which was the period that witnessed a high incidence of COVID-19 in South Africa and to some extent limiting the accessibility to different health facilities.www.nature.com/scientificreports/

RDT kit testing and data collection
RDT kit (ICT MALARIA DUAL Test-Malaria Pf/PAN antigen RDT, Cape Town, South Africa) was used for the testing.It was reported by the manufacturer to have been compared with microscopy based on 200 parasites/ ml and an International Laboratory evaluation against PCR showing a sensitivity of 96.8% and 96.3% for non P. falciparum and P. falciparum respectively and specificity of 99.7%.Following approved consent, the participant's finger was cleaned with an alcohol-based solution and allowed to air dry.A sterile lancet was used for the finger prick and 5 µl of blood was collected in the blood collection device supplied by the manufacturer and transferred to the cassette following which five drops of reaction buffer were placed in the large base well of the cassette and the outcome read in 15-30 min.The test outcomes were recorded as positive, negative or invalid and the used RDT kits were stored on-site at room temperature in dry pouches containing desiccants.This was followed by the administration of a short questionnaire containing the demographic data of the participants.The questionnaires were taken for statistical analysis while the RDT cassettes from these study sites were taken for further PCR testing in the laboratory.

DNA extraction and amplification
Each RDT cassette was opened and the nitrocellulose strip was carefully removed under sterile condition as recommended by an established protocol 51 .The proximal segment of the nitrocellulose strip was dissected and used for DNA extraction.The process of DNA extraction was done using the Zymo kit (Quick-DNA™ Miniprep-Plus Kit) based on the manufacturer's guidelines for nucleated blood samples with an elution volume of 50 µL.
The purity and concentrations of DNA were assessed using Nanodrop 2000.Samples were stored at minus 20 degrees centigrade until use.Nitrocellulose strips devoid of DNA were used for the control test.Thermo Electron® PX2 (HBPX2) thermal cycler was used for the primary and nested PCR procedure which was performed on the DNA extracts using published pairs of primers by Somé et al. 52 -(rPLUf 5′-TTA AAA TTG TTG CAG TTA AAACG-3' , rPLUr 5′-CCT GTT GTT GCC TTA AAC TTC-3′; nested PCR rFALf 5′-TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT -3′, rFALr 5′-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC -3′).Each reaction ran in total volume of 25 μl volume with 1 μl of forward and reverse primer in each case and 12.5 μl master mix added (1 × AmpliTaq Gold buffer, 1.5 mM MgCl 2, 0.25 mM dNTPs, 1 U of AmpliTaq Polymerase) to each reaction.Nuclease free water was 9.5 μl and 8.5 μl in the primary and the nested reactions respectively.2 μl of template DNA was added in primary while 1 μl of the PCR product of the first PCR was used as the template for the second PCR.The primary cycle of 35 cycles comprised an initial denaturation at 95 °C for 3 min, followed by final denaturation at 95 °C for 30 s, 53.1 °C 60 s and 72 °C for 60 s, and final elongation at 72 °C for 5 min.The nested PCR ran in 30 cycles and began with denaturation step at 95 °C for 5 min and extension at 95 °C for 30 s, 58.7 °C for 60 s, 72 °C for 60 s.The final elongation was at 72 °C for five minutes.The resultant amplicons from the secondary PCR were analysed on 1.5 (w/v) agarose gel electrophoresis at 70 V for 40 min and visualised under ultraviolet light using a Bio-RadChemiDoc™ MP System (Bio-Rad, US) with a baseline expected band of230bp.This was subsequently followed by Sanger sequencing of the PCR products with both forward and reverse primers at the Central Analytical Facility of the University of Stellenbosch, South Africa.

PCR confirmation of P. falciparum infection
In order to ensure that samples that will be used in the amplification of Pfhrp2, Pfhrp3 and their neighbouring genes have an adequate quality of genomic DNA; PCR amplification of the k1-allele of P. falciparum merozoite surface protein-1 (pfmsp1) was also performed on samples that tested positive for 18S rRNA gene and confrimed by Sanger's sequence.The processes of these gene amplifications were done using the method that was earlier described by Somé et al. 52 .The primers and annealing temperature used for both reactions are stated in Supplementary table 1.The primary cycle of 29 cycles comprised an initial denaturation at 94 °C for 30 s, 94 °C for 30 s, 53.1 °C for 60 s (annealing) and elongation at 68 °C for 60 s, and final elongation at 72 °C for 5 min.The nested PCR ran in 32 cycles and began with the denaturation step at 94 °C for 30 s, followed by 94 °C for 30 s, 68 °C for 5 min, 68 °C for 60 s, and final elongation was at 72 °C for five minutes.
For the Pfmsp1-k1, each of the primary and nested sample reaction was in a total volume of 25 μl comprising 2 μl of forward primer, 2 μl of reverse primer, 6.5 μl of nuclease free water and 12.5 μl Dreamtaq Green PCR Master mix (DreamTaq DNA polymerase, 2X Dreamtaq Green buffer, dATP, dCTP, dGTP and dTTP, 0.4 mM each, and 4 mM MgCl 2 , Thermo Fisher Scientific), 2 μl of genomic DNA was added in each reaction.The resultant PCR products were analysed on 1.5 (w/v) agarose gel electrophoresis at 70 V for 40 min and visualised under ultraviolet light using a Bio-RadChemiDoc™ MP System (Bio-Rad, US).

PCR detection of pfhrp2, pfhrp3 and their flanking genes
Samples that were RDT negative but amplified for both 18S r RNA gene and Pfmsp1were selected for detection of Pfhrp2 and Pfhrp3 deletion.The nested PCR protocol described by Abdallah et al. 39 was used for the amplification of exon1, the intron, and exon 2 of Pfhrp2 and Pfhrp3.Furthermore, the same protocol was used to assess the amplification of MAL7P1.230 and MAL7P1.228.The primers and annealing temperature used have been listed in Supplementary file, Table 1.Each reaction was performed in a total volume of 25 μl which consisted of forward primer (2μ), reverse primer (2μ), nuclease-free water (6.5 μl) and 12.5 μl Dreamtaq Green PCR Master mix (DreamTaq DNA polymerase, 2X Dreamtaq Green buffer, dATP, dCTP, dGTP and dTTP, 0.4 mM each, and 4 mM MgCl 2 , Thermo Fisher Scientific).The P. falciparum parasite strain 3D7 was used as a positive control for the PCR analyses of Pfhrp2, Pfhrp3 as well as their flanking genes.www.nature.com/scientificreports/

Sequence analysis and phylogeny
The forward and reverse sequences were edited using the BioEdit version 7.2 and these were submitted to the National Centre for Biotechnology Information (NCBI) and an accession number issued (OP341852-OP341871). Sequence similarity through Blastn analysis was performed and orthologous sequences were retrieved from the Gene Bank.Multiple sequence alignment of our sequences and those retrieved from the GenBank was carried out with Cluster-W program in MEGA version.Also, the best DNA model of the aligned sequences was evaluated using the AIC scores and phylogenetic tree based on Maximum Likelihood (ML) method was constructed with MEGA version 6.0 using Hasegawa-Kishino-Yano (HYK) model 53 with bootstrap of 1000.Babesia bigemina was used as an out-group to root the phylogenetic tree.

Statistical analysis
Statistical analysis was done to determine positivity outcome of tested RDT and PCR.Relationships between variables were assessed by Chi-square test or Fisher's exact test for categorical variables.The level of significance was set at α = 0.01 .The prevalence of the deleted Pfhrp2, Pfhrp3, and flanking gene was calculated by dividing the number of isolate in which there are deleted genes by the total number of isolates that were amplified by both 18S rRNA and msp-1.Statistical analyses were performed using the software package SPSSv.21.0.and Stata 14 (Stata Corp., College Station, TX, USA).

Ethical approval and consent to participate
The ethic for this study was approved by the Biomedical Research Ethics committee of the University of KwaZulu Natal South Africa with ethic approval number BREC/00001815/2020.